Polyester–Poly(Ethylene Glycol) Nanoparticles Loaded with the Pure Antiestrogen RU 58668: Physicochemical and Opsonization Properties Thibault Ameller, 1 Ve ´ ronique Marsaud, 1 Philippe Legrand, 1 Ruxandra Gref, 1 Gillian Barratt, 1 and Jack-Michel Renoir 1,2 Received March 4, 2003; accepted April 2, 2003 Purpose. The pure antiestrogen RU58668 (RU) was encapsulated within nanospheres (NS) and nanocapsules (NC) prepared from dif- ferent polyester copolymers with poly(ethylene glycol) (PEG) chains. The influence of their physicochemical properties on drug release in vitro and their susceptibility to opsonization were evaluated. Methods. RU-loaded PEG-bearing nanoparticles (NP) prepared by interfacial deposition of preformed polymer were characterized (size, zeta potential, percentage encapsulation and loading). In vitro release kinetics were studied in the presence of 10% fetal calf serum (FCS). Their opsonization in mouse serum was evaluated by silver staining of SDS-PAGE and Western blotting of desorbed proteins. Results. The NS were smaller than NC and had a zeta potential close to zero and a higher percentage of loading. RU release from NS in vitro was reduced as compared with the dissolution profile of free RU in a serum-containing medium. Decreased opsonin adsorption at the surface of pegylated NS was observed. Conclusion. Small nanoparticulate systems containing a high load of pure antiestrogen, showing reduced drug release, have been devel- oped. Among the six nanosphere preparations containing RU, two show a size below 200 nm, and two others undergo reduced protein adsorption in the presence of serum, compatible with increased per- sistence in the blood. KEY WORDS: nanospheres; nanocapsules; antiestrogen; opsonisa- tion. INTRODUCTION In 2001, the American Cancer Society estimated that ap- proximately 192,200 new cases of invasive breast cancers would be diagnosed among women in the United States. Moreover, according to the World Health Organization, more than 1.2 million people worldwide will be diagnosed with breast cancer this year. Tamoxifen (Tam) is considered as the antiestrogen of reference in the treatment of breast cancer. Nevertheless, the bioavailability of orally administered Tam citrate is very poor, and in addition, its agonist/antagonist activity leads to a number of undesirable effects. [see Mac- Gregor and Jordan (1) for a review], and resistance often occurs after long-term treatment. RU 58668 (RU), a new pure antiestrogen that was able to overcome Tam-induced resis- tance and to induce up to 30% disappearance of MCF-7 breast cancer tumors implanted in nude mice (2–4), appears to be a powerful alternative treatment for this disease. How- ever, because of the detrimental effects that could be pro- duced by a pure antiestrogen on the skeleton (inhibition of bone resorption) as well as on the cardiovascular and central nervous systems (decrease of the incidence of coronary heart disease and improvement of cognitive functions, respec- tively), it is necessary to develop a system capable of deliv- ering RU directly to the tumor. Many anticancer drugs, and in particular doxorubicin, have already been associated with particulate drug delivery systems such as liposomes, micelles, or polymeric nano- spheres in order to reduce their major side effects and to concentrate them in solid tumors [see Moghimi et al. (5) and references cited therein for a review]. When they are injected into the bloodstream, conventional drug delivery systems are rapidly recognized and removed from the circulation by the mononuclear phagocyte system (MPS) following opsoniza- tion by plasma proteins. The complement system, and in par- ticular the adsorption of C3 and its cleavage to C3b, is essen- tial for the recognition of foreign particles. Other proteins such as immunoglobulins (IgG) can contribute to the opso- nization process (6). An important strategy for avoiding opsonization, reduc- ing the rate of uptake by the MPS, and concentrating anti- cancer drugs within tumor tissues is modification of the sur- face properties of the drug carriers. For example, the pres- ence of hydrophilic chains such as poly(ethylene glycol) (PEG) at the surface of polymers or lipids drastically de- creases the protein adsorption when compared with unmodi- fied polymers or lipids, prolongs the circulation time of the encapsulated drug, and increases its probability of being taken up by tumor tissues after extravasation through discon- tinuous vascular endothelium (7). In this study, we chose to take advantage of the protein- rejecting properties of PEG by associating RU with nanopar- ticles (NP) prepared from biodegradable diblock copolymers in which polyester blocks consisting of such as poly(lactide) (PLA), poly(-caprolactone) (PCL), and poly(lactide–co- glycolide) (PLGA), which differ by their degradation rates and their hydrophobicity, were covalently coupled to PEG. Two different types of NP were studied: nanospheres, which are matrix systems with the drug entrapped within the poly- mer matrix and/or adsorbed on the surface, and nanocapsules, vesicular systems in which the drug is confined to an oily cavity surrounded by a thin polymeric membrane. After comparing the physicochemical properties of these two types of NP with different polyester cores, we determined the adsorption of specific opsonins onto their surface. The results obtained argue in favor of their prolonged persistence in the bloodstream, a prerequisite to tumor targeting. Be- cause no long circulating drug delivery system containing 1 UMR CNRS 8612, Pharmacologie Cellulaire et Mole ´ culaire, 92296 Cha ˆ tenay-Malabry, France. 2 To whom correspondence should be addressed. (e-mail: Michel. Renoir@cep.u-psud.fr) ABBREVIATIONS: C3, C3b, complement proteins C3, C3b; FCS, fetal calf serum; IgG, immunoglobulin G; MPS, mononuclear phago- cyte system; NC, nanocapsules; NP, nanoparticles; NS, nanospheres; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline, PCL, poly(-caprolactone); PEG, poly(ethylene glycol); PLA, poly(D,L-lactide); PLGA, poly(D,L-lactide–co-glycolide); SDS, so- dium dodecyl sulfate; RU, RU 58668 (11-[4-[5-[(4,4,5,5,5- pentafluoropentyl)sulfonyl]pentylo xy]phenyl]-estra-1,3,5(10)-triene- 3,17-diol; Tam, tamoxifen. Pharmaceutical Research, Vol. 20, No. 7, July 2003 (© 2003) Research Paper 1063 0724-8741/03/0700-1063/0 © 2003 Plenum Publishing Corporation